Concomitant knockdown resistance allele, L982W + F1534C, in Aedes aegypti has the potential to impose fitness costs without selection pressure.

The Aedes aegypti mosquito, is an arbovirus vector that can spread dengue, chikungunya, Zika, and yellow fever. Pyrethroids are widely used to control mosquitoes. The voltage-gated sodium channel (Vgsc) is the target of pyrethroids, and amino acid substitutions in this channel attenuate the effects of pyrethroids. This is known as knockdown resistance (kdr). Recently, we found that Ae. aegypti with concomitant Vgsc mutations L982W + F1534C exhibit extremely high levels of pyrethroid resistance. L982 is located in a highly conserved region of Vgsc in vertebrates and invertebrates. This study aimed to evaluate the viability of Ae. aegypti, with concomitant L982W + F1534C mutations in Vgsc. We crossed a resistant strain (FTWC) with a susceptible strain (SMK) and reared it up to 15 generations. We developed a rapid and convenient genotyping method using a fluorescent probe (Eprobe) to easily and accurately distinguish between three genotypes: wild-type and mutant homozygotes, and heterozygotes. As generations progressed, the proportion of wild-type homozygotes increased, and only 2.9% of mutant homozygotes were present at the 15th generation; the allele frequencies of L982W + F1534C showed a decreasing trend over generations. These observations show that these concomitant mutations have some fitness costs, suggesting that mosquitoes can potentially recover pyrethroid susceptibility over time without pyrethroid selection pressure in the field.

First detection of a Vssc allele V1016G conferring a high level of insecticide resistance in Aedes albopictus collected from Europe (Italy) and Asia (Vietnam), 2016: a new emerging threat to controlling arboviral diseases.

IntroductionAedes albopictus (Skuse) is an important vector of arboviral diseases, including dengue, chikungunya and Zika virus disease. Monitoring insecticide resistance and mechanisms by which the mosquito develops resistance is crucial to minimise disease transmission.AimTo determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions.MethodsWe sampled 33 populations of Ae. albopictus from Asia, Europe and South America, and tested these for susceptibility to permethrin, a pyrethroid insecticide. In resistant populations, the target site for pyrethroids, a voltage-sensitive sodium channel (Vssc) was genotyped. Three resistant sub-strains, each harbouring a resistance allele homozygously, were established and susceptibilities to three different pyrethroids (with and without a cytochrome P450 inhibitor) were assayed.ResultsMost populations of Ae. albopictus tested were highly susceptible to permethrin but a few from Italy and Vietnam (4/33), exhibited high-level resistance. Genotyping studies detected a knockdown resistance (kdr) allele V1016G in Vssc for the first time in Ae. albopictus. Two previously reported kdr alleles, F1534C and F1534S, were also detected. The bioassays indicated that the strain homozygous for the V1016G allele showed much greater levels of pyrethroid resistance than other strains harbouring F1534C or F1534S.ConclusionThe V1016G allele was detected in bothAsian and Italian Ae. albopictus populations, thus a spread of this allele beyond Italy in Europe cannot be ruled out. This study emphasises the necessity to frequently and regularly monitor the V1016G allele in Ae. albopictus, particularly where this mosquito species is the main vector of arboviruses.

Recent advances in the study of knockdown resistance mutations in Aedes mosquitoes with a focus on several remarkable mutations.

The Aedes mosquito, which transmits the dengue fever virus and other viruses, has acquired resistance to pyrethroid insecticides in a naturally selective manner. Massive use of insecticides has led to the worldwide expansion of resistant populations. The major factor in pyrethroid resistance is knockdown resistance (kdr) caused by amino acid mutation(s) in the voltage-gated sodium channel, which is the target site of this insecticide group. Some kdr mutations can lead to a dramatic increase in resistance, and multiple mutations can increase the level of pyrethroid resistance by 10 to several-hundred. In this review, we summarize the kdr identified in Aedes mosquitoes with a focus on the recent advances in the study of kdr.

Potential efficacy of olyset mosquito netting against Calliphora nigribarbis (Diptera: Calliphoridae) invasion into livestock barns.

Calliphora nigribarbis Vollenhoven is a possible mechanical transmitter of highly pathogenic avian influenza. Based on laboratory tests, we evaluated the efficacy of a long-lasting permethrin-treated mosquito netting, known as the Olyset net, for the prevention of this species entering livestock barns. Flies were trapped in Olyset net cages, and two statistics for knockdown and lethal efficacies were obtained. Median knockdown time in the cage (KT50) was estimated to be 341 s for females, and median lethal time after exposure to the mesh (LT50) was estimated to be 30 s and <15 s for females and males, respectively. These LT50s were faster than those measured for anesthetized stationary flies brought in contact with the Olyset net (> 120 s for both sexes),indicating that a fly's spontaneous contact with the Olyset net accelerates insecticide adhesion. The rate of permethrin adhesion to C. nigribarbis after its spontaneous contact with the Olyset net was estimated to be 3.7 ng/s for females, in reference to the 50% lethal dose (LD50) value (112 ng/female), which was obtained from the topical application bioassay of permethrin. The lethality exhibited after brief spontaneous contact with the Olyset net suggests its potential utility in poultry farms against C. nigribarbis invasion.

Discovery of super-insecticide-resistant dengue mosquitoes in Asia: Threats of concomitant knockdown resistance mutations.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

Common substitution mutation F348Y of acetylcholinesterase gene contributes to organophosphate and carbamate resistance in Cimex lectularius and C. hemipterus.

Bed bug control highly depends on insecticides with a limited number of modes of action, especially since the global prevalence of pyrethroid resistance. De facto insecticide options against bed bugs in Japan are acetylcholinesterase inhibitors (AChEis) that consist of organophosphates and carbamates. However, the status of AChEi resistance and the mechanisms involved have not been ascertained. An amino acid substitution mutation, F348Y (or F331Y in standard numbering), occurring at an acyl-binding site of the paralogous AChE gene (p-Ace), was identified among AChEi-resistant colonies of both common and tropical bed bugs (Cimex lectularius and C. hemipterus, respectively). This mutation was genetically associated with propoxur and fenitrothion resistance in F348Y-segregating colonies of C. hemipterus. Inhibition of heterologously expressed C. lectularius p-Ace with insecticides revealed that the sensitivities of F348Y-carrying AChE decreased by orders of 10- to more than 100-fold for diazoxon, carbaryl, fenitroxon, paraoxon, chlorpyrifos-methyl, malaoxon, azamethiphos, methyl-paraoxon, and propoxur. In contrast, the mutant AChE showed a slightly decreased degree of sensitivity for dichlorvos and almost unchanged sensitivity for metoxadiazone. Further studies are needed to ascertain whether the practical efficacies of dichlorvos and metoxadiazone are ensured against F348Y-carrying bed bugs and whether other resistance mechanisms are involved.

A first, naturally occurring substitution at the second pyrethroid receptor of voltage-gated sodium channel of Aedes aegypti.

BACKGROUND: Aedes aegypti is a remarkably effective mosquito vector of epidemiologically important arboviral diseases including dengue fever, yellow fever and Zika. The present spread of resistance against pyrethroids, the primary insecticides used for mosquito control, in global populations of this species is of great concern. The voltage-gated sodium channel (VGSC) in the nervous system is the known target site of pyrethroids in insects. Past studies have revealed several amino-acid substitutions in this channel that confer pyrethroid resistance, which are known as knockdown resistance (kdr) mutations. RESULTS: This study investigated a laboratory colony of Ae. aegypti, MCNaeg, established from larvae collected in Rio de Janeiro, Brazil in 2016. The MCNaeg colony showed strong resistance against pyrethroids without laboratory selection. Of the two VGSC gene haplotypes present within this colony, one harbored three known kdr mutations, V410L, V1016I, and F1534C, and the other harbored only the known F1534C mutation. In latter haplotype, we also found novel amino-acid substations including V253F. Previous molecular modeling and electrophysiological studies suggest that this residue serves a pyrethroid-sensing site in the second receptor, PyR2. Our genetical analysis showed that the haplotype harboring V253F and F1534C is associated with equal or slightly stronger resistance than the other triple kdr haplotype to both Type I and Type II pyrethroids. CONCLUSION: The novel substitution V253F is potentially involved in pyrethroid resistance in Ae. aegypti. Further studies are needed to elucidate the role of this substitution in the pyrethroid susceptibility of VGSC. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic analysis of Aedes aegypti captured at two international airports serving to the Greater Tokyo Area during 2012-2015.

The introduction of exotic disease vectors into a new habitat can drastically change the local epidemiological situation. During 2012-2015, larvae and an adult of the yellow-fever mosquito, Aedes aegypti, were captured alive at two international airports serving the Greater Tokyo Area, Japan. Because this species does not naturally distribute in this country, those mosquitoes were considered to be introduced from overseas via air-transportation. To infer the places of origin of those mosquitoes, we genotyped the 12 microsatellite loci for which the most comprehensive population genetic reference is currently available. Although clustering by Bayesian and multivariate methods both suggested that all those mosquitoes captured at the airports in Japan belonged to the Asia/Pacific populations, they were not clustered into a single cluster. Moreover, there was variation in mitochondrial cytochrome oxidase I gene (CoxI) haplotypes among mosquitoes collected in different incidents of discovery which indicated the existence of multiple maternal origins. We conclude there is little evidence to support the overwintering of Ae. aegypti at the airports; nevertheless, special attention is still needed to prevent the invasion of this prominent arbovirus vector.

RNA virome analysis of questing ticks from Hokuriku District, Japan, and the evolutionary dynamics of tick-borne phleboviruses.

Tick-borne viruses have emerged recently in many parts of the world, and the discoveries of novel tick-borne viruses have been accelerated by the development of high-throughput sequencing technology. In this study, a cost-efficient small benchtop next-generation sequencer, the Illumina MiniSeq, was used for the RNA virome analysis of questing ticks collected from Hokuriku District, Japan, and assessed for their potential utility in a tick-borne virus surveillance system. We detected two phleboviruses [Kabuto Mountain virus (KAMV) and Okutama tick virus (OKTV)], a coltivirus [Tarumizu tick virus (TarTV)], and a novel iflavirus [Hamaphysalis flava iflavirus (HfIFV)] from tick homogenates and/or cell culture supernatants after virus isolation processes. The number of sequence reads from KAMV and TarTV markedly increased when cell culture supernatants were used, indicating a successful isolation of these viruses. In contrast, OKTV and HfIFV were detected only in tick homogenates but not from cell culture supernatants, suggesting a failure to isolate these viruses. Furthermore, we performed genomic and phylogenetic analyzes of these detected viruses. OKTV and some phleboviruses discovered recently by NGS-based methods were probably deficient in the M genome segment, which are herein proposed as M segment-deficient phlebovirus (MdPV). A phylogenetic analysis of phleboviruses, including MdPV, suggested that Uukuniemi and Kaisodi group viruses and kabutoviruses evolved from an ancestral MdPV, which provides insights into the evolutionary dynamics of phleboviruses as emerging pathogens.

Prevalence of kdr-like mutations associated with pyrethroid resistance in human head louse populations in Japan.

Pyrethroid insecticides play very important roles in the control of pediculosis, which is caused by human head louse (Pediculus humanus capitis De Geer) worldwide. The development of head louse resistance to pyrethroid pediculicides has become a global issue during the last two decades. Pyrethroid resistance is associated with amino acid substitutions in the alpha-subunit of the para-sodium channel gene; these substitutions are termed kdr-like mutations. The frequencies of four mutations (D11E, M815I, T929I, and L932F) in the sodium channel gene were studied in human head louse colonies collected from Japan by using SNaPshot method. A total of 630 head lice collected from 282 infested people were analyzed, and it was found that 55 lice of 19 colonies homozygously or heterozygously possessed kdr-like genes, in which the four mutations existed concomitantly. This suggested that the quadruple mutant haplotype is a common feature of the kdr genes of pandemic head lice. The frequencies of the occurrence of resistant genes in the total individuals tested and resistant gene-carrying colonies were 8.7 (55/630) and 6.7% (19/263), respectively. Because the resistant gene was detected in the colonies collected from 11 of the 22 prefectures surveyed, it is speculated that resistant head lice are already spread extensively in Japan. This was the first large-scale survey of pyrethroid resistant head lice in Japan.

Spatial analysis and remote sensing for monitoring systems of Oncomelania nosophora following the eradication of schistosomiasis japonica in Yamanashi Prefecture, Japan.

In order to develop an inexpensive, simple, and accurate method of monitoring for the reemergence of schistosomiasis japonica in Yamanashi Prefecture, Japan, the distribution and habitation density of the intermediate host, Oncomelania nosophora, were spatially analyzed using geographic information systems. The 1967-1968 density distribution maps prepared by Yamanashi Prefecture and Nihei were digitized and geocoded. The habitats and population density of O. nosophora were estimated by referring to the data compiled by the Yamanashi Association for Schistosomiasis Control (1977). These earlier findings were compared with average population densities between 1996 and 2000 previously recorded (Nihei, N., Kajihara, N., Kirinoki, M., et al., Parasitol. Int., 52, 395-401, 2003 and Nihei, N., Kajihara, N., Kirinoki, M., et al., Parasitol. Int., 53, 199-205, 2004). A variance map was created to compare the spatial distribution maps of population density from each of the two periods of interest. The changes in distribution were remarkable and the map was found to be effective for future control. The most appropriate monitoring sites were chosen on the basis of the spatial population density maps and the variance map. Moreover, the paddy fields at risk were extracted using the normalized difference vegetation index value based on Advanced Land Observation Satellite images. The combination of this method with the global positioning system provides an inexpensive means of monitoring modern schistosomiasis endemic areas in Japan and also in China, the Philippines, and other countries as well, where the intermediate snail grows in paddy fields and marshlands under consistently wet conditions.

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations.

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline-MoNaS (Mosquito Na+ channel mutation Search)-which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.

PCR-based identification of Culex pipiens complex collected in Japan.

The Culex pipiens complex consists of vector mosquitoes that transmit important human pathogens. In this study we established a simplified method to distinguish three members of the Cx. pipiens complex, Cx. p. pallens Coquillet, Cx. p. form molestus Forskal, and Cx. quinquefasciatus Say, collected in Japan. Sequence analysis of the Drosophila Ace-orthologous acetylcholinesterase (Ace) gene (668 to 680 bp) revealed that a single polymorphic region characterizes each species. Based on this region, specific primers that distinguish Cx. p. form molestus (ACEpip2) and Cx. p. pallens (ACEpall2) were newly designed. Polymerase chain reactions were performed with the genomic DNA of Culex mosquitoes as the template, and these primers clearly distinguished two Culex spp. The accuracy of the designed primers was evaluated with 38 colonies of mosquito samples collected from 9 prefectures of Japan. The testing revealed that the distribution of anautogenous Cx. p. pipiens has not been confirmed in Japan. It also revealed that the male of Cx. p. pallens possesses an Ace gene haplotype that is highly similar to the sequence of Cx. quinquefasciatus. This improved method allows the evaluation of vector competence of Cx. p.molestus, which is the suspected vector of West Nile virus.

A mark-release-recapture study on dispersal and flight distance of Culex pipiens pallens in an urban area of Japan.

A mark-release-recapture of Culex pipiens pallens was conducted in an urban area of Japan during June 26-29, 2007. Larvae naturally occurring in rain gutters were collected and reared to adults in a laboratory. A total of 10,183 emerging female Cx. pipiens pallens of 4-8 days old were marked with fluorescent dye and released from one site. Recapture was made on 4 consecutive days using 41 Centers for Disease Control and Prevention traps with 1 kg of dry ice and human landing collection, and 121 marked females were recaptured. The overall recapture rate was 0.01. The mean distance traveled by the recaptured females was estimated as 470, 287, 326, and 517 m on days 1-4, respectively. The maximum flight distance of host-seeking Cx. pipiens pallens was estimated as 1,217 m based on the relationship between distance from the release site to the collection site and the total number of recaptures/traps. The population size of female Cx. pipiens pallens in the study area was estimated as 100,904 +/- 8,509. The size of operational area for the control of Cx. pipiens pallens in urban area is discussed.

Quantitative analysis of proliferation and excretion of Bartonella quintana in body lice, Pediculus humanus L.

Although body louse is a well-known vector of trench fever, the growth kinetics of Bartonella quintana in body lice has not been fully understood. We performed a quantitative analysis of bacterial multiplication rate. B. quintana started proliferation in body lice 4 days after ingestion and was constantly excreted in the feces for at least 3 weeks. The number of bacteria in feces reached the maximum 10(7)/louse per day on Day 15. The doubling time of B. quintana estimated from logistic regression formula was 21.3 hours. Scanning electron microscopy showed the presence of bacterial masses in feces. Immunofluorescent study using specific monoclonal antibody confirmed identification of B. quintana. Such an explosive multiplication rate and active excretion of B. quintana from the body lice could be related to epidemics of trench fever in developed countries.

Molecular characterization of two acetylcholinesterase cDNAs in Pediculus human lice.

Two cDNA sequences encoding Drosophila Ace-orthologous and -paralogous acetylcholinesterase precursors (AO- and AP-AChE precursors, respectively), were identified from the body louse, Pediculus humanus humanus L. In vitro inhibition studies with an insecticide-susceptible body louse strain exhibited a simplex inhibitory response of AChE. The I50 values of fenitroxon and carbaryl were estimated to be 2.2 and 1.9 microM for the susceptible lice, respectively. The mRNA level of AP-AChE gene was 3.1- and 9.3-fold higher than that of AO-AChE gene in the abdomen and the combined parts of the head and thorax, respectively, suggesting, due to its abundance, the potential significance of the AP-AChE isoform in Pediculus human lice in association with the efficacy of AChE-targeting pediculicides.

Insecticide resistance in potential vector mosquitoes for West Nile virus in Japan.

Culex pipiens complex is the significant vector mosquito of West Nile virus. To take stock of the current situation of insecticide susceptibilities and design an ideal mosquito control strategy, we collected Culex pipiens pallens Coquillet, Culex pipiens form molestus Forskal, and Culex quinquefasciatus Say from fields in Japan and conducted bioassays for five larvicides (fenitrothion, temephos, etofenprox, diflubenzuron, and pyriproxyfen) by using a larval dipping method. Among five insecticides tested, obvious reduced susceptibilities were observed for etofenprox, which is the only pyrethroid compound registered as a larvicide in Japan. Twenty-two of 56 colonies exhibited a >10% survival rate at the etofenprox concentration of 5.7 microg/ml, which is a 10 times higher concentration of the working solution. The LC50 of a colony collected from Fukuoka prefecture for etofenprox exceeded 60 microg/ml (resistance ratio >2,307), and this colony also exhibited cross-resistance to other pyrethroids, permethrin (299-fold) and phenothrin (1,200-fold). The insect growth regulators diflubenzuron and pyriproxyfen were found to be sufficiently effective enough to control Culex larvae present, but decreased sensitivities to these insecticides were slightly detected in some colonies of Cx. p. form molestus collected from urban areas. Several etofenprox-resistant colonies of Cx. p. form molestus exhibited simultaneously decreased susceptibilities to other insecticides, including temephos, diflubenzuron, and pyriproxyfen.

Predicting the Start of the Aedes albopictus (Diptera: Culicidae) Female Adult Biting Season Using the Spring Temperature in Japan.

Aedes albopictus (Skuse) (Diptera: Culicidae) is distributed widely and is common in much of Japan. In Japan, female adults begin to bite in between April and June, except in the southern subtropics where the mosquito has no dormant period. It is difficult to estimate the first Ae. albopictus biting day because it varies annually depending on the location. Over several years, we surveyed the mosquitoes at different locations that covered a range of warmer to cooler areas of Japan. We found an association between the timing of first biting day by Ae. albopictus and spring temperature. In spring months, the strongest correlation was found with mean April temperatures, followed by March. Based on these data, it may, therefore, be possible to apply a simple formula to predict the timing of the first biting day at various geographical locations in Japan. Forecasting maps were created using a simple prediction formula. We found that the first biting day for Ae. albopictus changed depending on early spring temperatures for each year. There was an approximate 20-d difference in first biting day between years with warmer and cooler springs. This prediction model will provide useful insight for planning and practice of Ae. albopictus control programs, targeting larvae and adults, in temperate regions globally.

Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.

Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.

Field and Laboratory Evaluations of the Efficacy of DEET Repellent against Ixodes Ticks.

The objective of this study was to clarify the efficacy of a currently available N,N-diethyl-m-toluamide (DEET) repellent against tick species in Japan. We performed 2 different field trials: "human trap," and "flag-dragging." In total, 482 ticks were collected from white flannel cloths in the field studies. The collected tick species were Ixodes persulcatus and I. ovatus, which accounted for 5.3% and 94.7% of the ticks in the human trap test and 31.4% and 68.6% in the flag-dragging test, respectively. The repellency levels of DEET-treated flannel cloths in the human trap and flag-dragging tests were 84.0% and 99.7%, respectively. The escape times for I. persulcatus and I. ovatus female adults from DEET-treated flannel cloths were determined. The median escape times for I. persulcatus and I. ovatus on DEET-treated flannel cloths were 48 s (95% confidence interval [CI]: 30-96) and 10 s (95% CI: 5-24), respectively. In contrast, many ticks remained on the untreated flannel cloths for 10 min after mounting. These results indicate that DEET repellents appear to prevent tick bites and that the use of DEET repellents against ticks is an effective personal protection measure.